(1) During the next year we will attempt to purify and characterize the endogenous inhibitor of colchicine binding previously identified in rat brain. Solubulization of the microsomal inhibitor will facilitate purification and will enable us to determine if the cytostolic and microsomal inhibitors are the same or different molecules. An attempt will also be made to further characterize the endogenous, heat stable factor which increases the colchicine binding capacity of tubulin. (2) The precise timing of assembly and disassembly of tubulin and HMW-MAPs into microtubules is being studied using double indirect immunofluorescence which permits HMW-MAPs and tubulin to be visualized in the same cell. Since some HMW-MAP staining persists in a filamentous distribution after complete depolymerization of microtubules it is important to determine whether the 10nm filaments of other cytoskeletal elements bind HMW-MAPs in situ. (3) We have developed hybridoma lines which produce antibodies to tubulin and, after cloning, the antibodies produced by these cells will be used as probes to study the various sites on tubulin which interact with other tubulin during assembly, with MAPs, and with small molecules such as GTP and colchicine. We are also planning to develop hybridomas which produce anti-MAP antibodies.